Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aÎ½ actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future. Human WAS-iPSCs (p.Phe35*) and gene corrected WAS-iPSCs (cWAS-iPSC) were differentiated into macrophages. WAS patient derived B-lymphocyte line ID00003 (p.Glu133Lys) and a wile-type B-lymphocyte line GM11518 were cultured using standard protocol. Total RNAs were extracted and been analyzed by RASL-seq.

Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aν actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future.

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